In Focus
Application of bacteriophages
Expert Round Table ParticipantsN: Rustam Aminov A , Jonathan Caplin B , Nina Chanishvili C , Aidan Coffey D , Ian Cooper E , Daniel De VosF , Jirí DoškarG , Ville-Petri FrimanH , I_ pek KurtbökeI , Roman Pantucek J , Jean-Paul PirnayF , Grégory ReschK , Christine RohdeL , Wilbert SybesmaM and Johannes WittmannL A
School of Medicine and Dentistry, University of Aberdeen, UK
B
School of Environment and Technology, University of Brighton, UK
C
Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia
D E F
Cork Institute of Technology, Department of Biological Sciences, Ireland
School of Pharmacy and Biomolecular Sciences, University of Brighton, UK
Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital, Brussels, Belgium
G
Masaryk University, Faculty of Science, Department of Experimental Biology, Brno, Czech Republic
H
University of York, Department of Biology, UK
I
University of the Sunshine Coast, GeneCology Research Centre and the Faculty of Science, Health, Education and Engineering, Qld, Australia
J
Masaryk University, Faculty of Science, Department of Experimental Biology, Brno, Czech Republic
K
University of Lausanne, Department of Fundamental Microbiology, Switzerland
L
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
M N
Nestec Ltd – Nestlé Research Center, Lausanne, Switzerland
Email:
[email protected]
The emergence of antibiotic-resistant bacteria and decrease
renewed interest in phage therapy is dictated by its advan-
in the discovery rate of novel antibiotics takes mankind
tages most importantly by their specificity against the
back to the ‘pre-antibiotic era’ and search for alternative
bacterial targets. This prevents complications such as
treatments. Bacteriophages have been one of promising
antibiotic-induced dysbiosis and secondary infections.
alternative agents which can be utilised for medicinal
This article is compiled by the participants of the Expert
and biological control purposes in agriculture and related
Round Table conference ‘Bacteriophages as tools for ther-
fields. The idea to treat bacterial infections with phages
apy, prophylaxis and diagnostics’ (19–21 October 2015) at
came out of the pioneering work of Félix d‘Hérelle but this
the Eliava Institute of Bacteriophage, Microbiology and
was overshadowed by the success of antibiotics. Recent
Virology, Tbilisi, Georgia. The first paper from the Round
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In Focus Table was published in the Biotechnology Journal1. This
resulted in increased survival of burn victims, most deaths are due
In Focus article expands from this paper and includes recent
to the wound sepsis or sepsis secondary to pneumonia. Animal
developments reported since then by the Expert Round
studies showed that bacteriophages could rescue mice and guinea
Table participants, including the implementation of the
pigs with infected burn wounds or bacteraemia. Ongoing studies
Nagoya Protocol for the applications of bacteriophages.
conducted following standard clinical trial guidelines and practices by ‘PhagoBurn’ (www.phagoburn.eu) will contribute towards
Antimicrobials are one of the most successful forms of therapy but their broad and often indiscriminate use resulted in a widespread antimicrobial resistance2. The annual death toll due to multidrugresistant bacterial infections is estimated at 23 000 in the US and 25 000 in Europe3,4. Complementary strategies are urgently needed, and bacteriophage therapy offers: *
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Specificity, and target-directed removal of pathogens via narrow spectrum, which do not affect beneficial commensals; Multiplication at infection sites, thus amplifying the local antimicrobial effects; Minimum, if any, side-effects; Resistance can be managed by introduction of new bacteriophages, which is faster and cheaper compared to new antibiotics; Bacteriophages are active against multidrug-resistant and biofilm-forming bacteria; Lytic bacteriophages may limit the evolution and spread of antimicrobial resistance5; Bacteriophages act in synergy with antibiotics; Phage CRISPR-Cas systems provide a new way to target antibioticresistant pathogens6.
generation of clinical level information related to the applications of phages. This phase I/II multi-centric, randomised, controlled and single-blinded clinical trial involves 15 burn units in France, Switzerland and Belgium and targets burn wounds infected by Escherichia coli or Pseudomonas aeruginosa. Manufacturing the investigational products that compline Good Manufacturing Practices (GMP) took 20 months and encountered poly-infection issues hampered the recruitment of patients8. However, the Phagoburn study has established new phage manufacturing approach that will encourage regulators to review their policies related to phage therapy8. Antagonistic bacterium-phage co-evolution is a dynamic process in which phage-resistant bacteria and infective bacteriophages are selected in turn. While emergence of bacteria resistant against challenging bacteriophages is a part of this coevolution, it could be problematic in therapy and it should be prevented. Interestingly,
Bacteriophage therapy was pioneered at the Eliava Institute in
while phage-resistant P. aeruginosa were readily selected in vitro
Tbilisi, Georgia (Figure 1), and the reader is referred to the
when challenged by the anti-P. aeruginosa phages used in
Historical Review article by Chanishvili and Sharp (2008)7 pub-
Phagoburn, such selection was not observed in a rat model of
lished in Microbiology Australia.
experimental endocarditis9. Accordingly, two resistant variants recovered in vitro showed >70% and >40% decreased infectivity,
Therapeutic application of bacteriophages and resistance: the case of Phagoburn Large burn wounds lead to immunosuppression, making burn patients susceptible to infections. Although medical advances have
explaining the failure to recover them from in vivo biopsies. These variants had lost lipopolysaccharide (LPS) and impaired pili, respectively, both structures being known as phage receptors10. This study illustrated that phage resistance can emerge at a very high cost in terms of virulence, possibly leading to in vivo survival for the bacterium. This observation, which is not new11, has clinical relevance and the phage resistance should be carefully evaluated in future clinical trials.
Bacteriophages for food hygiene and safety and environmental applications Bacteriophages have been used since the 1980s to control and eliminate bacterial contaminants from food surfaces, food-borne spoilage bacteria and bacteria causing gastrointestinal diseases12 as well as to decontaminate raw food. Due to their specificity, bacteriophages are attractive for sanitisation of ready-to-eat foods (RTE) such as milk, vegetables and meat products13. In 2007, the US Figure 1. Bacteriophage medicine sold to patients at the Eliava Institute’s Pharmacy. B
Department of Agriculture (USDA) approved bacteriophage products targeting Salmonella species and E. coli O157:H7. They are M I C R O B I O L O GY A U S T R A L I A
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In Focus designed as spray sanitisers to disinfect cattle hides prior to
commercialisation23. To ensure the efficiency of phage prepara-
slaughter to reduce pathogen contamination of meat14. In parallel,
tions, their effectiveness and host range towards currently circu-
the commercial product Agriphage was developed to control
lating pathogenic strains must be monitored. This might explain
black spot disease on tomato and pepper plants caused by Xantho-
why the phage preparations approved in the Russian Federation
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monas campestris and Pseudomonas syringae . Similarly, bacteriophages are also potentially useful as surface and environment decontaminants. Listeria phages (3.5 108 PFU/mL), for instance, were as effective as a 20-ppm solution of a quaternary ammonium compound (QAC) disinfectant for stainless steel de-
and Georgia are not static but are continuously updated to target newly emerging pathogenic strains24. Legislation to allow these updates is necessary to circumvent repeated registration procedures. On 5 July 2016, the Belgian Minister of Social Affairs and Public
contamination. Interestingly, synergism between different bacter-
Health has formally acknowledged that it is difficult to define the
iophages and phages-QAC was reported with bacteriophages being
status of therapeutic phage preparations: should they be consid-
unaffected by QAC at 50 ppm and up to 4 hours of contact time16.
ered as industrially-prepared medicinal products (subjected to constraints related to marketing authorisation) or as magistral
Agricultural applications of bacteriophages
preparations (prepared in pharmacies’ officina)25. Magistral pre-
Bacteriophage effects on target pathogens depend on the ecologi-
parations (compounded prescription drug products in the US) are
cal and environmental context such as abiotic environmental
made by a pharmacist from the constituent ingredients to meet
factors or surrounding microbial community. For example,
specific patient needs. On 26 October 2016, it was formally agreed
phage-mediated killing of pathogenic bacteria can be amplified in
that natural bacteriophages and their products, which are not fully
the presence of non-pathogenic bacteria that impose strong re-
compliant with the European Directive requirements for medicinal
source competition with the pathogen. More recently, it was shown
products for human use and for which there is no monograph in an
that the presence of antimicrobial producing Bacillus amylolique-
official pharmacopoeia, can be processed by a pharmacist as raw
faciens could shape the effect of bacteriophage selection on the
materials (active ingredients) in magistral preparations, providing
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plant pathogen Ralstonia solanacearum . In this case, the effect
compliance to several logical provisions.
was driven by evolutionary trade-off where evolving resistance to a phage led to increased susceptibility to antimicrobials produced by
virulence factors and reduced virulence in tomato in vivo18. Iden-
Bacteriophage application in the Access and Benefit Sharing (ABS) context: the Nagoya Protocol
tifying bacteriophages that impair pathogen virulence by binding to
To combat antibiotic resistances, there is urgent need to build
various surface structures (flagella, pili and LPS), could be impor-
up large phage collections against the pathogens like ESKAPE
tant for selecting therapeutic bacteriophages19.
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneu-
B. amyloliquefaciens. Similar evolutionary trade-offs can also lead to lowered expression of multiple important R. solanacearum
moniae, Acinetobacter baumannii, P. aeruginosa and EnteroWhen applied topically or orally to animals, bacteriophages will eventually become associated with the skin and wool/hair of animals. Thus, bacteriophages specific for animal pathogens could be isolated from wool20. These bacteriophages can reduce the number of bacteria associated with ’clumping’, and thus represent an option for agricultural practices as opposed to antibiotics. Similarly, bacteriophages have been recovered from the skin of healthy humans21 or when they were successfully incorporated into fibers used for human clothing22.
bacteriaceae). However, culture collections holding and offering quality-checked authenticated bacteriophages in the sense of phage banks are confronted with two constraints. First, there are no requirements for authors by journals to deposit bacteriophages with public repositories before publishing, which differs from agreed procedures for their bacterial hosts26. The second issue that should be considered is the current development of rules for legal handling of bioresources that of course includes the bacteriophages. On 12 October 2014, the Nagoya Protocol https://www. cbd.int/abs/ has been implemented in several countries that ratified
Current hurdles and regulatory status of bacteriophages
the Convention on Biological Diversity (CBD) https://www.cbd.int/. These laws deal with sampling, the accession and distribution
Bacteriophages are not currently classified in medicinal legislation,
of all genetic resources including microorganisms regarding the
since they are neither living nor chemical agents. Therefore,
ABS. One of the reasons for the ratification of the protocol is
it is complicated to regulate and perform clinical trials and
protecting biodiversity under national sovereignty to prevent
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In Focus ‘biopiracy’ and to restrict access. All microbiologists who are sam-
7.
Chanishvili, N. and Sharp, R. (2008) Bacteriophage therapy: experience from the Eliava Institute, Georgia. Microbiol. Aust. 29, 96–101.
8.
Servick, K. (2016) DRUG DEVELOPMENT. Beleaguered phage therapy trial presses on. Science 352, 1506. doi:10.1126/science.352.6293.1506
9.
Oechslin, F. et al. (2016) Synergistic interaction between phage therapy and antibiotics clears Pseudomonas aeruginosa infection in endocarditis and reduces virulence. J. Infect. Dis. jiw632. doi:10.1093/infdis/jiw632
pling or distributing bioresources must be aware of these restrictions and should refer to their respective national regulations. National regulations might differ in each country and failure to comply with might result in legal consequences. For further information please see the DSMZ website at https://www.dsmz.de/deposit/nagoya-protocol.html.
Conclusions and future perspectives As already stated by Skurnik and Strauch (2006) a decade ago27, the therapeutic use of bacteriophages, possibly combined with antibiotics, is a promising therapy option. Safe and controlled use of bacteriophage therapy will however, require as detailed information as possible on the properties and behaviour of specific phagebacterium systems, in vitro and especially in vivo. Susceptibility of bacterial pathogens in vivo to bacteriophages is still not completely understood and requires dedicated (pre-)clinical research on more phage-bacterium systems. The requirements for quality and safety in bacteriophage production and application have been defined and communicated28–30. Natural resources will need to be utilised further to isolate many more bacteriophages to build-up large phage collections to fight the antibiotic crisis. These efforts will then be translated into cooperation across borders and continents that will be regulated by the Nagoya Protocol to some extent. Therefore, facilitative regulations governing therapeutic use of bacteriophages should be implemented to counter antibiotic resistance on a global scale. Bacteriophage application obviously have significant potential to bridge human and veterinary medicine and bring effective solutions to antibiotic resistance problems as pointed out in this article.
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